Mutationally activated ABL tyrosine kinases are causative in many leukemias. ABL kinase inhibitors have been useful in treating chronic myeloid leukemia (CML) with a BCR-ABL1 fusion, demonstrating the value of oncoprotein-directed therapeutics. However, some leukemias with activated ABL do not respond to these inhibitors and some CML patients that respond, subsequently become resistant. Resistance, and disease relapse, most often results from BCR-ABL1 kinase domain mutations that maintain kinase activity but prevent inhibitor binding. To understand the mechanism of leukemogenesis, and to explore potential therapeutic targets in kinase inhibitor-resistant leukemias, we examined a physiological activator of ABL kinases. RIN1 directly binds ABL1 and stimulates kinase activity through de-repression of the autoinhibited enzyme. RIN1 strongly stimulates ABL kinases in vitro and in vivo. In addition, RIN1 binds to and enhances the catalytic, transforming and leukemogenic properties of BCR-ABL1. This demonstrates that the tyrosine kinase activity of BCR-ABL1, while elevated and constitutive relative to ABL1, is still responsive to stimulation by RIN1. Deletion of RIN1 blocks transformation of bone marrow cells by BCR-ABL1. Transformation is rescued by re-introduction of RIN1, indicating that this is a cell autonomous phenotype. Silencing of RIN1 in leukemia cells reduced cellular phospho-tyrosine levels and sensitized cells to the ABL inhibitor imatinib. BCR- ABL1T315I, a drug resistant mutant found in CML patients, was also dependent on RIN1 for transformation. The activation of ABL kinases by RIN1 suggests an alternative approach to kinase inhibition: disrupting the interaction with a positive regulator. Because BCR-ABL1 is dependent on RIN1 for full transformation, this represents a unique point of vulnerability that could be exploited to treat kinase inhibitor-resistant leukemias. Combining drugs that inhibit BCR-ABL1 activation by RIN1 with standard ABL kinase inhibitors could provide therapy that is more efficacious and less prone to the development of resistance and disease relapse. We have created an assay that quantifies the collaboration of RIN1 and ABL1. The assay incorporates a novel application of time-resolved fluorescence resonance energy transfer (TR-FRET) and was validated in a high throughput format with robust characteristics (Z'>0.5). An exceptionally diverse chemical library will be included in a screen for compounds that inhibit the interaction of RIN1 and ABL1. Secondary screens will be used to eliminate false positives and to prioritize inhibitors that can block transformation by BCR-ABL1. This proposal addresses a fundamental question in signal transduction (how are non-receptor tyrosine kinases regulated?), with clear disease relevance (how do leukemogenic ABL kinases transform cells?) and immediate clinical implications (how can resistance to standard kinase inhibitors be prevented or circumvented?). The compounds identified in this screen will be invaluable tools for understanding the biochemistry of normal and oncogenic ABL proteins, and may also serve as leads to new therapeutics. PUBLIC HEALTH RELEVANCE: The proposed research uses a novel assay to identify inhibitors of an ABL tyrosine kinase regulator with a direct role in leukemogenesis. The assay, which includes an innovative use of fluorescence technologies, will be used in a high throughput screen for compounds that block leukemia-associated kinases independently of standard inhibitors. Compounds in this category should be invaluable tools for understanding the biochemistry underlying this disease. Derivatives of such inhibitors may eventually be developed into effective therapeutics against the increasing number of drug resistant leukemias and might synergize with standard kinase inhibitors to reduce the incidence of drug resistance in these and other tyrosine kinase-dependent cancers.